重组乳酸乳球菌不同途径发送shRNA抗肿瘤的研究

Anti-tumor study of shRNA transmitted by different routes of recombinant Lactococcus lactis

[目的]比较重组乳酸乳球菌不同途径发送HPV16E7 shRNA的抗肿瘤效果。[方法]通过实时定量PCR技术筛选出抑制Si Ha细胞中E7基因表达的shRNA片段并构建至乳酸乳球菌质粒p MG36e-RFP中。PCR鉴定获得重组乳酸乳球菌正确后分别通过滴鼻、静脉注射和瘤内注射途径发送到C57BL/6小鼠体内后,检测荷瘤小鼠的生存周期及体内肿瘤的生长大小。[结果]成功构建针对HPV16E7的shRNA干扰表达的重组乳酸乳球菌,肿瘤注射后32 d,鼻腔发送途径组小鼠肿瘤大小约为6. 49±1. 60 cm3,静脉注射组肿瘤大小为4. 49±1. 16 cm3,瘤内注射组肿瘤大小为1. 89±0. 52 cm3,至肿瘤注射60d时,瘤内注射组有3只存活。[结论]瘤内和静脉注射表达shRNA重组乳酸乳球菌均能抑制小鼠体内肿瘤的生长,其中瘤内途径优于静脉注射。

[Objective] Comparing the anti-tumor effect of HPV16 E7 shRNA by transmitted different routes of Lactococcus lactis.[Method]The shRNA fragment which inhibited the expression of E7 gene in Si Ha cells was screened by real-time quantitative PCR and constructed into the recombinant plasmid p MG36 e-RFP of Lactococcus lactis. After being identified by sequencing,recombinant Lactococcus lactis was sent to C57 BL/6 mice by intranasal,intravenous and intratumoral injection to detect the survival cycle of tumor-bearing mice and the growth of tumors in vivo. [Result]The shRNA interference expression vector targeting HPV16 E7 was successfully constructed and recombinant Lactococcus lactis was obtained by Electrical transduction. After 32 days of tumor injection,the tumor size of the nasal delivery group was about 6. 49 ± 1. 60 cm3,the tumor size of the intravenous group was 4. 49 ± 1. 16 cm3,and the tumor size of the intratumoral delivery group was 1. 89 ± 0. 52 cm3. And there were still 3 mice surviving in the intratumoral injection group by the time of tumor injection for 60 days.[Conclusion]Intratumoral and intravenous injection of shRNA-recombinant Lactococcus lactis inhibited tumor growth in mice,and the intratumoral route was superior to intravenous injection.